Methods and Compositions To Enhance Efficiency Of Nuclear Transfer/Cloning

ABSTRACT

The present invention provides compositions and methods for increasing the success of assisted reproductive technology (ART). Specifically, the inventions described herein increase the survival rate of manipulated embryos for increasing post implantation numbers of viable offspring. In particular, the present invention provides for compositions and methods for allowing further embryonic development and increasing rates of embryonic maturation, such as increasing cleavage rate, TE numbers, and blastocyte formation of in vitro fertilized and nuclear transfer embryos in media comprising follistatin, thereby providing for increased survival of fertilized and manipulated embryos leading to increased numbers of live offspring from in vitro fertilized and implanted nuclear transfer embryos. Further provided are diagnostic kits for determining transplantation potential.

FIELD OF THE INVENTION

The present invention provides compositions and methods for increasingthe success of assisted reproductive technology (ART). Specifically, theinventions described herein increase the survival rate of manipulatedembryos for increasing post implantation numbers of viable offspring. Inparticular, the present invention provides for compositions and methodsfor allowing further embryonic development and increasing rates ofembryonic maturation, such as increasing cleavage rate, TE numbers, andblastocyte formation of in vitro fertilized and nuclear transfer embryosin media comprising follistatin, thereby providing for increasedsurvival of fertilized and manipulated embryos leading to increasednumbers of live offspring from in vitro fertilized and implanted nucleartransfer embryos. Further provided are diagnostic kits for determiningtransplantation potential.

BACKGROUND OF THE INVENTION

Poor oocyte competence contributes to infertility in humans andlivestock species. Two well-defined bovine models of oocyte competenceare the prepubertal oocyte and time to first cleavage models. Bovineembryos produced in vitro from oocytes harvested from prepubertalanimals show reduced development to the blastocyst stage (Revel et al.,1995, J. Reprod. Fertil. 103:115-120; Damiani et al., 1996, Mol. Repro.Dev. 45:521-534; all of which are herein incorporated by reference) andtransfer of in vitro (Khatir et al., 1998, Theriogenology 50:1201-1210)and in vivo produced bovine embryos (Armstrong et al., 2001,Theriogenology 55:1303-1322; all of which are herein incorporated byreference) derived from oocytes of prepubertal animals results inreduced pregnancy success. Furthermore, a higher proportion of bovineembryos that cleave early (e.g., 30 h post fertilization), rather thanlate (e.g., 36 h post fertilization) reach the blastocyst stage (Planteet al., 1994, Mol. Repro. Dev. 39:375-383; Lonergan et al., 1999, J.Repro. Fertil. 117:159-167; all of which are herein incorporated byreference).

During the initial cleavage divisions post-fertilization, embryonicdevelopment is supported by maternal mRNAs and proteins synthesized andstored during oogenesis which are critical for the interval betweenfertilization and the maternal-embryonic transition when transcriptionalactivity of the embryonic genome becomes robust at the 8-16 cell stagein cattle (Telford et al., 1990, Mol. Repro. Dev. 26:90-100; De Sousa etal., 1998, Mol. Repro. Dev. 51:112-121; all of which are hereinincorporated by reference). Thus, time to first cleavage of early bovineembryos is likely significantly influenced by differences in oocytederived mRNA and proteins. However, the cloning/nuclear transfertechnology for propagation of valuable livestock species, generation oftransgenic animals, therapeutic cloning and research purposes iscurrently limited by low efficiency.

As such, what are needed are compositions and methods for increasing theefficiency of assisted reproductive technologies. In particularincreased rates of blastocyte and trophectoderm formation are needed forcultured embryos.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for increasingthe success of assisted reproductive technology (ART). Specifically, theinventions described herein increase the survival rate of manipulatedembryos for increasing post implantation numbers of viable offspring. Inparticular, the present invention provides for compositions and methodsfor allowing further embryonic development and increasing rates ofembryonic maturation, such as increasing cleavage rate, TE numbers, andblastocyte formation of in vitro fertilized and nuclear transfer embryosin media comprising follistatin, thereby providing for increasedsurvival of fertilized and manipulated embryos leading to increasednumbers of live offspring from in vitro fertilized and implanted nucleartransfer embryos. Further provided are diagnostic kits for determiningtransplantation potential.

In one embodiment, the present invention contemplates a culture mediumfor in vitro culture of embryos comprising follistatin, wherein thedevelopment and survival of said embryos is enhanced when grown in saidculture media compared to growth in culture media without follistatin.In one embodiment, the follistatin is present at a concentration fromabout 1 ng/ml to about 20 ng/ml. In one embodiment, the follistatin ispresent at a concentration of about 10 ng/ml. In one embodiment, themethod further comprises an embryo, wherein said embryo is selected fromthe group consisting of in vitro fertilization embryos, nuclear transferembryos, cloned embryos, noncloned embryos, embryos for assistedreproductive techniques. In one embodiment, the embryos are mammalianembryos.

In one embodiment, the present invention contemplates a kit comprising aculture medium and mammalian embryos. In one embodiment, the kit furthercomprises a sheet of instructions.

In one embodiment, the present invention contemplates a method ofincreasing the survival of embryos, comprising, a) providing, i) anembryo, ii) a culture medium, wherein said culture medium comprisesfollistatin, b) culturing said embryo in a culture medium wherein thesurvival of said embryo is increased compared to survival of an embryonot grown in said media. In one embodiment, the embryo is an in vitrofertilization embryo. In one embodiment, the nuclear transfer embryo isa nuclear transfer embryo. In one embodiment, the nuclear transferembryo comprises genetic material obtained from a somatic cell. In oneembodiment, the follistatin is present at a concentration from about 1ng/ml to about 20 ng/ml. In one embodiment, the follistatin is presentat a concentration of about 10 ng/ml. In one embodiment, the increasedsurvival of said embryo is increasing the number of trophectoderm cells.In one embodiment, the follistatin is a human recombinant follistatin.In one embodiment, the method further comprises obtaining a biopsy fromsaid embryo. In one embodiment, the method further comprises c)determining the amount of follistatin expression in said embryo.

In one embodiment, the present invention contemplates a method ofgenerating stem cells comprising: a) providing, i) an embryo, ii) aculture medium, wherein said culture medium comprises follistatin, b)culturing said embryo in a culture medium to provide cultured embryoniccells, c) generating stem cells from said cultured embryonic cells. Inone embodiment, the culture of said embryo in said media generates anincreased number of stem cells from said embryo as compared to thenumber of stem cells generated from an embryo not grown in said culturemedium. In one embodiment, the method further comprises step d)harvesting said stem cells. In one embodiment, the follistatin ispresent at a concentration from about 1 ng/ml to about 20 ng/ml.

Other illustrative embodiments of the invention are described below. Thepresent invention is not limited to these embodiments.

In some embodiments, the present invention provides compositions andmethods for maintenance/growth of nuclear transfer cloned embryoswherein said maintenance/growth favors the survival of said embryos, thedevelopment of trophectoderm cells in a blastocyst, and increases thenumbers of live offspring from said embryos. As such, methods andcompositions of the present invention provide a wide range ofapplications, including but not limited to applications to thecommercial cloning industry for livestock and animal biotechnologyindustries, human regenerative medicine and research applicationsincluding noncloned and cloned embryos.

In some embodiments, the present invention provides for the developmentof culture medium comprising, for example, a compound of interest andthe development of tools/diagnostics to select eggs with high levels ofendogenous follistatin. In some embodiments, the compound of interest isfollistatin. In some embodiments, follistatin is added to alreadyexisting culture media as defined in U.S. Pat. Nos. 5,096,822,5,563,059, and 5,693,534, all incorporated by reference herein in theirentireties. In some embodiments, the present invention provides for theculture of nuclear transfer embryos in the above-mentioned culture mediathereby increasing the viability of said embryos and offspring resultingfrom said embryos.

In some embodiments, the present invention provides methods forselecting oocytes for nuclear transfer of genetic material for nucleartransfer cloning comprising measuring the level of endogenousfollistatin in said oocytes and selecting for oocytes with increasedlevels of endogenous follistatin for nuclear transfer of geneticmaterial (e.g., DNA).

In some embodiments, the present invention provides for culture mediafor in vitro culture of embryos, in some embodiments nuclear transferembryos, comprising follistatin, wherein the development and survival ofsaid embryos is enhanced when grown in said culture media compared togrowth in culture media without follistatin. In some embodiments thefollistatin is in the culture media at a concentration of about 1 ng/mlto about 20 ng/ml. In some embodiments the follistatin is in the culturemedia at a concentration of about 10 ng/ml. In some embodiments, theembryo cultured in the culture medium supplemented with follistatin arederived from groups including but not limited to in vitro fertilizationembryos, nuclear transfer embryos, cloned embryos, noncloned embryos,embryos for assisted reproductive techniques, and the like.

In some embodiments, the present invention provides methods forincreasing the survival of nuclear transfer embryos comprising providingembryos comprising nuclear transferred genetic material, such as from invitro fertilization, nuclear transfer cloning, somatic cells, and thelike, growing and maintaining the embryos in a culture media comprisingfollistatin of concentrations of about 1 ng/ml to 20 ng/ml wherein thesurvival of the embryos is increased compared to survival of embryos notgrown in the follistatin supplemented media. In some embodiments, thenuclear transferred genetic material is somatic cell genetic material.

In further embodiments, the present invention provides methods forincreasing the number of trophectoderm cells in a nuclear transferembryo derived blastocyst comprising providing embryos comprisingnuclear transferred genetic material and growing the embryos infollistatin supplements culture media at a concentration of about 1ng/ml to about 20 ng/ml, and increasing the number of trophectodermcells when the embryos are grown in the follistatin supplemented mediacompared to embryos grown in non-follistatin supplemented media. In someembodiments, the cultured embryos are derived from in vitrofertilization or nuclear transfer cloning. In some embodiments, thenuclear transferred genetic material in the embryos is somatic cellmaterial.

In some embodiments, the present invention provides methods for thegeneration of stem cells comprising providing somatic cell nucleartransfer embryos, culturing the embryos in a culture media comprisingfollistatin at a concentration of, for example, about 1 ng/ml to about20 ng/ml and deriving the stem cells from the cultured embryonic cells.In some embodiments, the culture of the somatic cell nuclear transferembryos in follistatin-supplemented media generates an increased numberof stem cells compared to embryos grown in non-follistatin supplementedmedia. In some embodiments, the methods of the present invention furtherprovide for the harvesting of the stem cells from embryos grown infollistatin-supplemented media.

In some embodiments, the present invention provides methods forincreasing transplantation potential, comprising, providing, an embryo,a medium comprising follistatin, incubating said embryo in said medium,determining the amount of expressed follistatin. In one embodiment, theembryo provides a biopsy for determining the amount of follistatin. Inone embodiment, the biopsy is evaluated for the amount of expressedfollistatin. In one embodiment, amount of expressed follistatin isfollistatin mRNA. In one embodiment, the amount of expressed follistatinis follistatin protein. In one embodiment, the amount of expressedfollistatin is determined using an antifollistatin antibody.

In some embodiments, the present inventions provide a diagnostic kit fordetermining embryonic transplantation potential. In one embodiment, thekit comprises detection reagents for determining embryonictransplantation potential, including but not limited to reagents fordetecting follistatin mRNA expression, an antifollistatin antibody, andthe like.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an exemplary demonstration of an effect of follistatinsupplementation during the first 72 h of in vitro culture of bovineembryos on the time to first cleavage. Effect of follistatin treatmenton proportion of embryos that reached the 2-cell stage (A) within 30 hpost insemination (early cleaving), and (B) from 30-36 h postinsemination (late cleaving), Values are expressed as mean±SEM of thedata collected from six replicates. Values with different superscriptsacross treatments indicate significant differences (P<0.05).

FIG. 2 shows an exemplary demonstration of an effect of follistatinsupplementation during the first 72 h of in vitro culture of bovineembryos on development to the blastocyst stage (day 7). The values areexpressed as mean±SEM of the data collected from six replicates. Valueswith different superscripts across treatments indicate significantdifferences (P<0.05).

FIG. 3 shows an exemplary demonstration of an effect of follistatinsupplementation on rate of development of early versus late cleavingbovine embryos to the blastocyst stage in vitro. Effects of exogenousfollistatin on (A) proportion of early cleaving embryos reaching theblastocysts stage and (B) proportion of late cleaving embryos reachingthe blastocyst stage are depicted. The values are expressed as mean±SEMof the data collected from four replicates. Values with differentsuperscripts across treatments indicate significant differences(P<0.05).

FIG. 4 shows an exemplary demonstration of an effect of follistatinsupplementation during the first 72 h of in vitro culture of bovineembryos on day 7-blastocyst cell allocation. Effect of exogenousfollistatin supplementation on (A) the number of inner cell mass (ICM)cells, (B) number of trophectoderm (TE) cells and (C) total cell numbersare depicted. The total number of blastocysts examined for stainingafter 0, 1, 10 and 100 ng/ml follistatin treatment was 14, 22, 27 and15, respectively. The values are expressed as mean±SEM of the datacollected from four replicates. Values with different superscriptsacross treatments indicate significant differences (P<0.05).

FIG. 5 shows an exemplary demonstration of an effect of follistatinsupplementation during first 72 h of in vitro culture of bovine embryoson % ICM cells and TE cells in day 7 blastocyst stage embryos. Effect ofexogenous follistatin supplementation on (A) the proportion of ICM cellsversus total cells and (B) the proportion of TE cells versus total cellsare depicted. The values are expressed as mean i SEM of the datacollected from four replicates. Values with different superscriptsacross treatments indicate significant differences (P<0.05).

FIG. 6 shows an exemplary demonstration of an effect of follistatinsupplementation during first 72 h of in vitro culture of bovine embryoson the proportion of embryos reaching blastocyst stage (day 7) withdifferent ICM/total cell ratios (ICM ratio) reflective of embryoquality. Effects of exogenous follistatin supplementation on proportionsof day 7 blastocysts with (A) ICM ratio of 20-40%, (B) ICM ratio of40-60% and (C) ICM ratio of >60% are depicted. The values are expressedas mean±SEM of the data collected from four replicates. Values withdifferent superscripts across treatments indicate significantdifferences (P<0.05).

FIG. 7 shows an exemplary demonstration of A) an effect of follistatinshort interfering RNA (siRNA) knockdown of follistatin mRNA and proteinin bovine in vitro fertilized embryos in vitro and B) an effect ofmicroinjection of follistatin siRNA on follistatin protein abundance in16-cell embryos in vitro. The results are expressed as mean±SEM of thedata collected from four replicates. Values with different superscriptsacross treatments indicate significant differences (P<0.05).

FIG. 8 shows an exemplary demonstration of an effect of microinjectionof follistatin siRNA on development of in vitro fertilized bovineembryos; A) early cleaving embryos, B) 8-16 cell embryos, and C)blastocysts. The results are expressed as mean±SEM of the data collectedfrom four replicates. Values with different superscripts acrosstreatments indicate significant differences (P<0.05).

FIG. 9 shows an exemplary demonstration of the effect of follistatintreatment on development of nuclear transfer embryos to the blastocyststage. The results are expressed as mean±SEM of the data collected fromfour replicates. Values with different superscripts across treatmentsindicate significant differences (P<0.05).

FIG. 10 shows an exemplary demonstration of an effect of follistatintreatment on cell allocation in nuclear transfer blastocysts. The effectof follistatin treatment on number of trophectoderm cells is depicted.The number of blastocysts examined for staining were 22 (parthenogeneticembryo control), 17 (0 ng/ml follistatin), 23 (1 ng/ml follistatin), 26(10 ng/ml follistatin) and 15 (100 ng/ml follistatin). The values areexpressed as mean±SEM of the data collected from four replicates. Valueswith different superscripts across treatments indicate significantdifferences (P<0.05).

FIG. 11 shows an exemplary demonstration of an effect of follistatinsupplementation during first 72 h of in vitro culture of bovine embryoson % ICM cells and TE cells in day 7 blastocyst stage embryos generatedvia nuclear transfer. Effect of exogenous follistatin supplementation on(A) the proportion of ICM cells versus total cells and (B) theproportion of TE cells versus total cells are depicted. The values areexpressed as mean±SEM of the data collected from four replicates. Valueswith different superscripts across treatments indicate significantdifferences (P<0.05).

FIG. 12 shows an exemplary demonstration of an effect of follistatinsupplementation during first 72 h of in vitro culture of bovine nucleartransfer embryos on the proportion of embryos reaching blastocyst stage(day 7) with different ICM/total cell ratios (ICM ratio) reflective ofembryo quality. No significant effect of follistatin treatment onproportions of day 7 nuclear transfer blastocysts with ICM ratio of20-40% or 40-60% was observed. The values are expressed as mean±SEM ofthe data collected from four replicates. Values with differentsuperscripts across treatments indicate significant differences(P<0.05).

FIG. 13 shows an exemplary demonstration of an effect of follistatinablation (siRNA microinjection) and (or) replacement (follistatinsupplementation) on bovine embryonic development. Presumptive zygoteswere microinjected (16-18 h post-fertilization) with approximately 20 plof follistatin siRNA or served as uninjected controls. Uninjected orinjected presumptive zygotes were cultured serum free in KSOM mediumsupplemented with 0.3% BSA and with or without 10 ng/ml follistatin (FS)maximally effective dose determined in previous studies; 25-30presumptive zygotes per group). The 8-16 cell stage embryos were thenseparated and cultured in fresh KSOM medium supplemented with 0.3% BSAand 10% FBS in absence of exogenous follistatin until d 7. Effect offollistatin ablation and (or) replacement on (Top) proportion of embryosdeveloping to the 8-16 cell stage and (Bottom) proportion of embryosdeveloping to blastocyst stage. Data are expressed as mean±SEM from fourreplicates. Values with different superscripts across treatmentsindicate significant differences (P<0.05).

FIG. 14 shows an exemplary demonstration of an effect of follistatinablation (siRNA microinjection) and (or) replacement [follistatin (FS)supplementation] on cell allocation within bovine blastocysts (d 7).Number of inner cell mass (ICM), trophectoderm (TE) and total cellnumbers were determined by cell counts on differentially stained embryos(Machaty et al., 1998; herein incorporated by reference). Effect offollistatin ablation and (or) replacement on (Top) number of ICM cells,(Middle) number of TE cells and (Bottom) total cell numbers arerecorded. Data are expressed as mean±SEM from four replicates. Valueswith different superscripts across treatments indicate significantdifferences (P<0.05).

FIG. 15 shows A) an exemplary demonstration of an effect of follistatinablation (siRNA microinjection) and (or) replacement [follistatin (FS)supplementation] on mRNA abundance for the inner cell mass marker Nanog(top) and trophectoderm (TE) cell marker CDX-2 (bottom) in bovineblastocysts. Blastocysts were harvested at d 7 post-fertilization (n=4pools of 2 blastocysts each per treatment) and subjected to RNAisolation and quantitative real-time RT-PCR analysis of mRNA abundancefor Nanog and CDX-2. Data were normalized relative to abundance ofendogenous control (18S rRNA) and are shown as mean±SEM. Means withoutcommon superscripts in each panel are significantly different (P<0.05).B) an exemplary schematic diagram of where Nanog and CDX-2 influencecell fate in a developing embryo (Duranthon, et al., Reproduction (2008)135 141-150, FIG. 2; herein incorporated by reference).

FIG. 16 shows an exemplary demonstration of an effect of follistatinsupplementation (10 ng/ml) during initial 48 h of in vitro culture ofrhesus monkey embryos on (Top) % cleavage at 30 h post insemination and(Bottom) % development to the blastocyst stage (determined on d 8).(a,b; P<0.05).

DEFINITIONS

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, livestock, vertebrates suchas rodents, non-human primates, ovines, bovines, ruminants, lagomorphs,porcines, caprines, equines, canines, felines, aves, etc.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

The term “transgene” as used herein refers to a foreign gene that isplaced into an organism by, for example, introducing the foreign geneinto newly fertilized eggs or early embryos, thereby producing, forexample, a “transgenic animal”. The term “foreign gene” refers to anynucleic acid (e.g., gene sequence) that is introduced into the genome ofan animal by experimental manipulations and may include gene sequencesfound in that animal so long as the introduced gene does not reside inthe same location, as does the naturally occurring gene.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, transformed celllines, finite cell lines (e.g., non-transformed cells), and any othercell population maintained in vitro. Cells cultured in the presentapplication include oocytes, embryos and embryo derived cell masses.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNA,or a polypeptide or its precursor (e.g., proinsulin). A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the polypeptide are retained. The term “portion”when used in reference to a gene refers to fragments of that gene. Thefragments may range in size from a few nucleotides to the entire genesequence minus one nucleotide.

The term “transgene” as used herein refers to a foreign, heterologous,or autologous gene that is placed into a cell or an organism byintroducing the gene into newly fertilized eggs or early embryos, suchas a gene encoding an siRNA of the present inventions. The term “foreigngene” refers to any nucleic acid (e.g., gene sequence) that isintroduced into the genome of an animal by experimental manipulationsand may include gene sequences found in that animal so long as theintroduced gene does not reside in the same location as does thenaturally-occurring gene.

The term “autologous gene” is intended to encompass variants (e.g.,polymorphisms or mutants) of the naturally occurring gene. The termtransgene thus encompasses the replacement of the naturally occurringgene with a variant form of the gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in an embryo, a host cell may be a zygote.

As used herein, the term “somatic cell” in general refers to any diploidmammalian cell, such as a fibroblast, and the like, with the exceptionof a fertilized egg (i.e. zygote).

As used herein, the term “gamete” or “germ cell” in reference to a cellrefers to cell consisting of a single (haploid) set of chromosomes.Specifically, a gamete is selected from a sperm cell (a spermatocyte orspermatozoa) and an egg cell (oocyte), in other words a “reproductivecell.”

As used herein, the term “ploidy” refers to the number of sets ofchromosomes within a cell or an organism. For example, haploid means oneset of chromosomes and diploid means two sets of chromosomes.

As used herein, the term “cell differentiation” in general refers to aprogressive restriction of developmental potential and increasingspecialization of function of cells that takes place during progressivestages of development of the embryo which leads to the formation ofspecialized cells, tissues, and organs.

As used herein, the term “totipotent” in general refers to an embryoniccell capable of giving rise to all types of differentiated cell found inthat organism. In other words, a single totipotent cell could, bydivision, reproduce a whole organism, for example, a zygote.

As used herein, the term “pluripotent” refers to a cell capable ofmaturing or develop in any of several ways.

As used herein, the term “pluripotent stem cell” refers to a cell whosedescendants are capable of developing into many different types of cellsor tissues in the body.

As used herein, the term “meiosis” refers to a reductive cell divisionwhich results in daughter cells containing one copy of each of thechromosomes of the parent, such as the process that produces a spermcell and an egg cell. The entire meiotic process involves two separatedivisions (Meiosis I (Reduction) and Meiosis II (Division)). Meiosis Iand II are both divided into prophase, metaphase, anaphase, andtelophase, such as prophase I, prophase II, and the like.

As used herein, the term “mitosis” refers to a process whereby a cellnucleus divides into two daughter nuclei, each having the same geneticcomponent as the parent cell; in other words mitosis refers to nucleardivision plus cytokinesis, and produces two identical daughter cellsduring prophase, prometaphase, metaphase, anaphase, and telophase.

As used herein, the term “cell cycle” refers to a cell while engaged inmetabolic activity and performing its preparation for mitosis(prometaphase, metaphase, anaphase, and telophase), where the cell cycleis divided into interphase G1 (GAP 1)-S (DNA synthesis)-G2 (GAP 2)-M(mitotic) stages.

As used herein, the term “cleavage” in reference to an embryo refers toearly embryo cleavage from one cell to at least a 2-cell stage.

As used herein, the term “enucleated” refers to a cell from which thenucleus was removed, such as an oocyte used for nuclear transfer toproduce a cloned animal from a nucleus from a somatic cell, such as adifferentiated cell.

As used herein, the term “stem cell” refers to a Relativelyundifferentiated cells of the same lineage (family type) that retain theability to divide and cycle throughout postnatal life to provide cellsthat can become specialized

As used herein, the term “stem cell” in reference to an embryo, as in an“embryonic stem cell” refers to totipotent cell.

As used herein, the term “medium” in reference to a liquid or gelatinoussubstance containing nutrients for culturing a cell or tissue (such asan embryo) refers to a singular form while media refers to a plural formof the substance.

As used herein, the term “stem cell” refers to a generalized “mother” or“parental” cell that has pluripotency, i.e. daughter cells ordescendants may specialize in different functions, such as anundifferentiated mesenchymal cell that is a progenitor (stem cell) forboth red and white blood cells.

The term “antisense” refers to a deoxyribonucleotide sequence whosesequence of deoxyribonucleotide residues is in reverse 5′ to 3′orientation in relation to the sequence of deoxyribonucleotide residuesin a sense strand of a DNA duplex. A “sense strand” of a DNA duplexrefers to a strand in a DNA duplex which is transcribed by a cell in itsnatural state into a “sense mRNA.” Thus an “antisense” sequence is asequence having the same sequence as the non-coding strand in a DNAduplex. The term “antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA;many antisense RNAs block the expression of a target gene by interferingwith the processing, transport and/or translation of its primarytranscript, for example mRNA. The complementarity of an anti sense RNAmay be with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence. In addition, antisense RNA may contain regions of ribozymesequences that increase the efficacy of anti sense RNA to block geneexpression. “Ribozyme” refers to a catalytic RNA and includessequence-specific endoribonucleases. “Anti sense inhibition” refers tothe production of anti sense RNA transcripts capable of preventing theexpression of the target protein, or of preventing the function of atarget RNA.

The term “siRNAs” refers to short interfering RNAs. In some embodiments,siRNAs comprise a duplex, or double-stranded region, where each strandof the double stranded region is about 18 to about 25 nucleotides long;the double stranded region can be as short as 16, and as long as 29,base pairs long, where the length is determined by the anti sensestrand. Often siRNAs contain from about two to four unpaired nucleotidesat the 3′ end of each strand. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants. At least one strand of theduplex or double-stranded region of a siRNA is substantially homologousto or substantially complementary to a target RNA molecule. The strandcomplementary to a target RNA molecule is the “antisense strand;” thestrand homologous to the target RNA molecule is the “sense strand,” andis also complementary to the siRNA antisense strand. One strand of thedouble stranded region need not be the exact length of the oppositestrand; thus, one strand may have at least one fewer nucleotides thanthe opposite complementary strand, resulting in a “bubble” or at leastone unmatched base in the opposite strand. One strand of the doublestranded region need not be exactly complementary to the oppositestrand; thus, the strand, preferably the sense strand, may have at leastone mismatched base-pair.

siRNAs may also contain additional sequences; non-limiting examples ofsuch sequences include linking sequences, or loops, which connect thetwo strands of the duplex region. This form of siRNAs may be referred to“si-like RNA,” “short hairpin siRNA,” where the short refers to theduplex region of the siRNA, or “hairpin siRNA.” Additional non-limitingexamples of additional sequences present in siRNAs include stem andother folded structures. The additional sequences may or may not haveknown functions; non-limiting examples of such functions includeincreasing stability of an siRNA molecule, or providing a cellulardestination signal.

The term “target RNA molecule” refers to an RNA molecule to which atleast one strand of the short double-stranded region of an siRNA iscomplementary. Typically, when such complementarity is about 100%, thesiRNA is able to silence or inhibit expression of the target RNAmolecule. Although it is believed that processed mRNA is a target ofsiRNA, the present invention is not limited to any particularhypothesis, and such hypotheses are not necessary to practice thepresent invention. Thus, it is contemplated that other RNA molecules mayalso be targets of siRNA. Such targets include unprocessed mRNA,ribosomal RNA, and viral RNA genomes. The term “ds siRNA” refers to asiRNA molecule which comprises two separate unlinked strands of RNAwhich form a duplex structure, such that the siRNA molecule comprisestwo RNA polynucleotides.

The term “enhancing the function” when used in reference to an siRNAmolecule means that the effectiveness of an siRNA molecule in silencinggene expression is increased. Such enhancements include but are notlimited to increased rates of formation of an siRNA molecule, decreasedsusceptibility to degradation, and increased transport throughout thecell. An increased rate of formation might result from a transcriptwhich possesses sequences which enhance folding or the formation of aduplex strand.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing expression, or inhibition of expression, of gene expressionby siRNAs. It is the process of sequence-specific, post-transcriptionalgene silencing in animals and plants, initiated by siRNA that ishomologous in its duplex region to the sequence of the silenced gene orthat is complementary in its duplex region to the transcriptionalproduct of the silenced gene. The gene may be endogenous or exogenous tothe organism, present integrated into a chromosome or present in atransfection vector which is not integrated into the genome. Theexpression of the silenced gene is either completely or partiallyinhibited. The term “transfection” as used herein refers to theintroduction of foreign DNA into eukaryotic cells. Transfection may beaccomplished by a variety of means known to the art including calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, electroporation, microinjection,liposome fusion, lipofection, protoplast fusion, retroviral infection,and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes.

The term “transient transfectant” refers to cells that have taken upforeign DNA but have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973];herein incorporated by reference), has been modified by several groupsto optimize conditions for particular types of cells. The art is wellaware of these numerous modifications.

The term “assisted reproductive technologies” or “ART” refers to anyinfertility treatment that uses advanced technology to combine sperm andeggs outside the body in a laboratory, including any infertilitytreatment that uses advanced technology to combine sperm and eggsoutside the body in a laboratory in order to increase fertility.Examples include but are not limited to “ICSI” or “IntracytoplasmicSperm Injection” referring to a technique that allows reproductivespecialists to isolate sperm from a male with which to fertilize eggs invitro. Once fertilization has taken place, the embryo is allowed todevelop for a few days and is then implanted into a uterus at theappropriate time of the reproductive cycle, “ZIFT” or “ZygoteIntrafallopian Transfer” refers to fertilization taking place before theegg is placed inside of fallopian tubes. A sample of sperm is mixed withharvested eggs. Once fertilization has taken place (creating a zygote)the fertilized egg is implanted surgically into fallopian tubes.

As used herein, the term “nuclear transfer” is a form of cloning. Thesteps involve removing the DNA from an oocyte (e.g., unfertilized egg),and injecting a nucleus containing the DNA from the individual to becloned. Typically, the survival of nuclear transferred derived embryosin non-human mammals (e.g., sheep, bovine, etc.) is very inefficient andembryo viability is very low. The term “somatic cell nuclear transfer”refers to the transfer of DNA from a somatic cell or the entire cellitself (e.g., heart cell, skin cell, nerve cell, etc.) into the emptyoocyte. The present invention increases the survival of nuclear transfercloning embryos through development resulting in an increase in numberof blastocyst stage embryos and viable offspring or stem cells (e.g.,somatic cell nuclear transfer cloning).

As used herein, the term “infertility” refers to a state of beinginfertile, for example, not being able to conceive, either in vivo or invitro, or not being able to support development in order to deliver,either naturally or by cesarean, a live child. As used herein, the term“fertility” refers to a state of being fertile, for example, being ableto conceive, either in vivo or in vitro, or being able to supportdevelopment in order to deliver, either naturally or by cesarean, a livechild.

As used herein, the term “IVM” or “in-vitro maturation” refers to amaturation of an immature oocyte in vitro attempting to duplicate thenatural process that occurs within the follicle in vivo.

As used herein, the term “IVF” or “in-vitro fertilization” refers tofertilization of an oocytes with a sperm outside of an organism. IVF mayalso refer to a technique, whereby oocytes and spermatozoa are mixed inthe laboratory to achieve fertilization.

As used herein, the term “IVC” or “in-vitro culture” refers to anincubation of fertilized oocytes (zygotes) in the laboratory through theprocess of cleavage, typically up to the blastocyst stage ofdevelopment.

As used herein, the term “IVP” or “in-vitro production” refers to acombined process of in-vitro maturation, in-vitro fertilization andin-vitro culture whereby embryos are produced in the laboratory (i.e.IVP=IVM+IVF+IVC).

As used herein, the term “intracytoplasmic sperm injection” refers to amicromanipulation procedure whereby a single spermatozoon is inserteddirectly into the cytoplasm of the oocyte to achieve fertilizationduring IVF.

As used herein, the term “blastocyst” refers to a thin-walled hollowstructure in early embryonic development that contains a cluster ofcells called the inner cell mass from which the embryo arises.

As used herein, the term “inner cell mass” or “trophoblast” refers to apart of the blastocyst that will give rise to the embryo proper, asopposed to the extra-embryonic membranes.

As used herein, the term “implantation” refers to a process whereby theblastocyst stage embryo burrows into the lining of the uterus, orendometrium, to establish a pregnancy.

As used herein, the term “transplantation potential” refers to thecapability of an embryo to develop normally to term (birth) followingembryonic transplantation, such as by using ART in combination with thepresent inventions, i.e. a fertilized embryo, a nuclear transplantembryo, and the like.

As used herein, the term “instructions” or “sheet of instructions” as in“instructions for using said kit. In one embodiment for addingfollistatin” includes instructions for using the reagents contained inthe kit for adding variant (i.e. interfering RNA) and wild typefollistatin, such as polypeptides, nucleotides, antibodies, receptorantibodies, and the like.

In one embodiment for “detecting follistatin” such as a diagnostic kitprovided for determining transplantation potential, includesinstructions for using the reagents contained in the kit for thedetection of variant and wild type follistatin nucleotides.

As used herein, “follistatin” refers to any portion of a follistatinmolecule, including a nucleic acid and a protein, or portion or fragmentthereof, for providing the enhancement of embryonic development as shownherein. Thus, a follistatin may be a molecule from any species of livingorganism, and refer to any portion of that molecule provided that ashowing of enhancement of embryonic development is provided. Thisenhancement of embryonic development is contemplated to lead to anincrease in efficiency for ART of any species, for providing ES cellsand any contemplated use of the present inventions related to culturingembryos in follistatin.

In some embodiments, the instructions further comprise the statement ofintended use required by the United States (U.S) Food and DrugAdministration (FDA) in labeling in vitro diagnostic products. The FDAclassifies in vitro diagnostics as medical devices and requires thatthey be approved through the 510(k) procedure. Information required inan application under 510(k) includes: 1) The in vitro diagnostic productname, including the trade or proprietary name, the common or usual name,and the classification name of the device; 2) The intended use of theproduct; 3) The establishment registration number, if applicable, of theowner or operator submitting the 510(k) submission; the class in whichthe in vitro diagnostic product was placed under section 513 of theFederal Food, Drug, and Cosmetic Act, if known, its appropriate panel,or, if the owner or operator determines that the device has not beenclassified under such section, a statement of that determination and thebasis for the determination that the in vitro diagnostic product is notso classified; 4) Proposed labels, labeling and advertisementssufficient to describe the in vitro diagnostic product, its intendeduse, and directions for use. Where applicable, photographs orengineering drawings should be supplied; 5) A statement indicating thatthe device is similar to and/or different from other in vitro diagnosticproducts of comparable type in commercial distribution in the U.S.,accompanied by data to support the statement; 6) A 510(k) summary of thesafety and effectiveness data upon which the substantial equivalencedetermination is based; or a statement that the 510(k) safety andeffectiveness information supporting the FDA finding of substantialequivalence will be made available to any person within 30 days of awritten request; 7) A statement that the submitter believes, to the bestof their knowledge, that all data and information submitted in thepremarket notification are truthful and accurate and that no materialfact has been omitted; 8) Any additional information regarding the invitro diagnostic product requested that is necessary for the FDA to makea substantial equivalency determination. Additional information isavailable at the Internet web page of the United States Food and DrugAdministration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for increasingthe success of assisted reproductive technology (ART). Specifically, theinventions described herein increase the survival rate of manipulatedembryos for increasing post implantation numbers of viable offspring. Inparticular, the present invention provides for compositions and methodsfor allowing further embryonic development and increasing rates ofembryonic maturation, such as increasing cleavage rate, TE numbers, andblastocyte formation of in vitro fertilized and nuclear transfer embryosin media comprising follistatin, thereby providing for increasedsurvival of fertilized and manipulated embryos leading to increasednumbers of live offspring from in vitro fertilized and implanted nucleartransfer embryos. Further provided are diagnostic kits for determiningtransplantation potential.

The inventions provide compositions and methods for increasing theproportion of nuclear transfer embryos that develop to the blastocyststage of development thereby enhancing the number of trophectoderm cellsand the quality of embryos such that an increase in live births fromsaid embryos is contemplated. An increase in blastocyst rate for nucleartransfer embryos would also increase the efficiency of therapeuticcloning and generation of embryonic stem cells for cell therapy.

It was previously demonstrated that follistatin is a marker of eggquality in cattle. The effects of endogenous follistatin on earlyembryonic development of in vitro fertilized bovine embryos were testedand stimulatory effects on development to the blastocyst stage andnumber of trophectoderm cells/embryo quality were observed. Given thestimulatory effects on blastocyst development and embryo quality(trophectoderm cells) observed and the observation that the number oftrophectoderm cells and quality of nuclear transfer embryos is believedresponsible for the less than 5% efficiency of generation of liveoffspring, the effects of follistatin on development of nuclear transferembryos to the blastocyst stage and numbers of trophectoderm cells insuch embryos was tested.

Results indicate a pronounced stimulatory effect of follistatin ondevelopment of nuclear transfer embryos to the blastocyst stage,indicating follistatin treatment results in more transferable embryos.Results also indicate that follistatin treatment significantly enhancesembryo quality (number of trophectoderm cells) and results in a greaterproportion of live offspring born after transfer of nuclear transferembryos.

In developing embodiments of the present invention, differences in RNAtranscript profiles in oocytes and 2-cell stage bovine embryosassociated with oocyte competence were performed. Experimental resultsindicated that follistatin mRNA is greater in germinal vesicle stageoocytes collected from prepubertal versus adult animals (Patel et al.,2007, Repro. 133:95-106; herein incorporated by reference). Furthermore,follistatin mRNA abundance is greater in early cleaving two cell bovineembryos (collected prior to the maternal zygotic transition andinitiation of significant transcription from the embryonic genome) thanin their late cleaving counterparts (Patel et al., 2007; hereinincorporated by reference). It was contemplated that follistatin had astimulatory role in early embryonic development. Example 2 demonstratesthe effects of exogenous follistatin treatment during the first 72 h ofin vitro culture (to 16-cell stage) of bovine embryos on time to firstcleavage, development to the blastocyst stage and blastocyst cellallocation (embryo quality). To evaluate the requirement of endogenousfollistatin for bovine early embryogenesis, experiments were performedas reported in Example 2 thereby demonstrating the effect of follistatinmRNA knockdown (via microinjection of follistatin siRNA) on time tofirst cleavage and successful development of bovine embryos to the 8-16cell and blastocyst stages. Given the robust effects of follistatintreatment on blastocyst development and cell allocation (in favor oftrophectoderm) of in vitro fertilized embryos and demonstratedrequirement of follistatin for early embryogenesis, it was demonstratedthat follistatin treatment enhances the efficiency of nuclear transferby determining the effects of follistatin supplementation on cellallocation and blastocyst development of nuclear transfer embryos asseen in Example 4.

Certain illustrative embodiments of the invention are described below.The present invention is not limited to these embodiments.

In some embodiments, the present invention provides compositions andmethods for identifying and selecting oocytes and embryos with increasedlevels of endogenous follistatin for nuclear transfer and/or in vitrofertilization procedures. For example, polar bodies that are removedduring nuclear transfer cloning procedures are assayed for follistatin.Alternatively, cells of a multi-celled embryo cells as a four-cell,eight-cell or sixteen-cell embryo can be biopsied and analyzed. Assaysfor follistatin include, but are not limited to, enzyme linkedimmunosorbent assays (ELISA) (Evans et al., 1998, J. Endo. 156:275-282;herein incorporated by reference). For identifying and selectingpost-transfer embryos for increased levels of follistatin for continuedculture, one cell from an embryo, for example, is harvested (e.g.,embryo biopsy by removing one cell from an embryonic cell mass) andassayed for follistatin levels. When levels of follistatin are increasedin either the oocyte or the post-transfer embryo, the nuclear transferor continued culture, respectively would be carried out. In someembodiments, an additional application of the present invention is themeasurement of follistatin in embryo culture media and using it as aguide (positive marker) for selecting embryos to transfer. This hasapplication to human assisted reproductive technologies (ART) (e.g. invitro fertilization).

In some embodiments, oocytes are harvested and placed in culture mediacomprising at least 1 ng/ml, at least 5 ng/ml, 10 ng/ml, at least 20ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least100 ng/ml follistatin, thereby increasing endogenous levels offollistatin in the oocytes prior to nuclear transfer and/or in vitrofertilization. In some embodiments, follistatin is microinjecteddirectly into an oocyte using techniques known by those skilled in theart, thereby increasing levels of endogenous follistatin in said embryo.

In some embodiments, follistatin is used to supplement media generallyused to culture embryos. For example, in some embodiments, follistatinis used to supplement potassium simplex oxidized medium (KSOM), a basemedia for nuclear transfer oocyte and embryo culture. KSOM is typicallysupplemented with serum (e.g., fetal bovine serum (FBS), human serum,horse serum (HS), etc.) prior to use and may be supplemented with anamino acid cocktail and other components deemed necessary for cellsurvival. In some embodiments, no serum is present during the time offollistatin supplementation. Exemplary formulations useful inembodiments of the present invention for KSOM can be found in, forexample, U.S. Pat. No. 5,541,081, Erbach et al., 1994, Biol. of Repro.50:1027-1033, and Lawitts and Biggers, 1993, Meth. in Enzym.225:153-165; all incorporated herein by reference in their entireties.KSOM base media is commercially available from, for example, MilliporeSpecialty Media. Millipore Specialty Media also provides new KSOMformulations for use as base media for oocyte and embryo culture.However, methods and compositions of the present invention are notlimited by the type of culture media used and any type of media and/orsupplementation to any media is contemplated, including media systemsthat are serum free.

KSOM media components typically include, for example, sodium chloride,potassium chloride, potassium hydrophosphate, magnesium sulfate,lactate, glucose, pyruvate, sodium bicarbonate, calcium chloride,glutamine, EDTA, source of mammalian serum (e.g., bovine, fetal bovine,human) and penicillin/streptomycin (e.g., for inhibition of bacterialcontamination). PH is typically adjusted to around 7.4. KSOM media isused to culture oocytes and embryos through the blastocyst stage priorto implantation. The KSOM used in the present invention is typicallysupplemented with BSA (0.3%) or FBS (10%), however follistatin is addedduring the serum free component of culture. A skilled artisan willunderstand what is deemed necessary for media supplementation for anyparticular culture system, not limited to KSOM. In the presentapplication, the KSOM media is further supplemented with follistatin at1 ng/ml, 10 ng/ml or 100 ng/ml in serum free conditions.

For in vitro fertilization there can be from one to more than oneculture and maintenance media used. For example, Gordon in U.S. Pat. No.5,512,476 (incorporated herein by reference in its entirety) describesthe formulation for a culture and fertilization media comprising salts,amino acids, vitamins, pyruvate, glucose, and other components.Alternatively, Gardner and Lane (U.S. Pat. No. 6,838,235; incorporatedherein in its entirety) describe the formulations and usage of an 1)oocyte retrieval and handling medium, an 2) oocyte maturation medium, a3) fertilization medium, 4) several embryonic development media, an 5)embryo transfer media, and 6) a media for cryopreservation. Otherformulations for IVF media useful as media in embodiments of the presentinvention can be found at, for example, U.S. Pat. Nos. 5,627,066,5,691,194, 6,281,013, and 6,585,982 and in The Handbook of in vitroFertilization, Eds. Trouson and Gardner, Informa Health Care Publ.,2000, and In vitro Fertilization and Embryo Culture: A Manual of BasicTechniques, Ed. Wolf, Springer Publ., 1988 (all incorporated herein intheir entireties). In either a single or a multi-media system, it iscontemplated that one or more of the IVF media are supplemented withfollistatin at a concentration of, for example, about 1 ng/ml up toabout 20 ng/ml.

Methods and compositions for the culture of stem cells are found in, forexample, U.S. Pat. Nos. 7,220,584, 7,217,569, 7,148,062, 7,029,913,7,005,252, 6,200,806, 5,843,780 and 6,328,764; all incorporated hereinby reference in their entireties.

In some embodiments, compositions of the present invention comprisenuclear transferred embryos, superovulated embryos, and/or in vitrofertilized embryos cultured in culture media comprising at least 1ng/ml, at least 5 ng/ml, at least 10 ng/ml, at least 20 ng/ml, at least30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 100 ng/mlfollistatin, thereby increasing survival of the embryos. In someembodiments, the culture media comprises preferably 1.0-20 ng/mlfollistatin, more preferably 1-10 ng/ml follistatin. In someembodiments, culture of the nuclear transfer embryos in culture media aspreviously exemplified provides for differential blastocyst cellallocation. In some embodiments, culture of the nuclear transfer embryosin culture media as previously exemplified further provides forincreased numbers of trophectoderm cells in blastocysts in proportion toinner cell mass cells, compared to embryos not grown in follistatincontaining media. In some embodiments, follistatin treatment of nucleartransfer embryos increases, for example, their development to blastocyststage and enhances, for example, blastocyst cell allocation in favor oftrophectoderm cells, wherein increased numbers of trophectoderm cellsenhances, for example, placentation, and wherein, for example, increasedplacentation increases the number of live birth offspring from saidnuclear transfer embryos grown in follistatin containing media (e.g., atleast 1 ng/ml, at least 5 ng/ml, at least 10 ng/ml, at least 20 ng/ml,etc.).

In some embodiments, the present invention provides in vitrofertilization methods that use media supplemented with follistatin.Protocols for performing in vitro fertilization (IVF) can be found at,for example, U.S. Pat. Nos. 4,589,402, 4,725,579 and in The Handbook ofin vitro Fertilization, Eds. Trouson and Gardner, Informa Health CarePubl., 2000, and In vitro Fertilization and Embryo Culture: A Manual ofBasic Techniques, Ed. Wolf, Springer Publ., 1988; all incorporatedherein in their entireties. There are several issues associated withsuccess in performing IVF. Those issues include, but are not limited to,zona pellucida hardening that leads to decrease in sperm penetration,temperature of fertilization and maintenance of eggs, sperm and embryos,pH, the occurrence of volatile organic compounds found in laboratory airthat can harm the process, and other environmental factors.

An exemplary protocol for human in vitro fertilization can be dividedinto several stages; 1) oocyte stimulation, 2) oocyte retrieval, 3) invitro fertilization, 4) embryo transfer, and 5) post transfer.Basically, ovulation stimulation is induced in a female such that thefemale starts developing multiple follicles on the ovaries.Gonadotropins such as follicle stimulating hormone or analogues thereofare injected to initiate the developmental process. Spontaneousovulation is blocked using injections of gonadotropin releasing hormone(GnRH) antagonist that blocks the surge of luteinizing hormone. Whenfollicular maturation is adequate, human chorionic gonadotropin (hCG) isgiven, and oocytes are retrieved from the female prior to approximately36 hours after injection of the hCG. Follicles are aspirated from theovaries and prepared in the laboratory for fertilization with the sperm.The oocytes are incubated with the sperm in culture media as exemplifiedabove for about 18 hours. Fertilization is complete with the observationof two pronuclei in the embryo. However, if fertilization is notrealized, for example if sperm count is low, one or more sperm can beinjected into the oocyte using intracytoplasmic sperm injections (ICSI).The new embryo(s) is transferred to growth media as exemplified aboveat, for example, day 3 prior to the blastocyst stage, at 5 day(blastocyst stage), and sometimes embryos are transferred at the 6-8cell stage. In some embodiments, the growth media comprises follistatinas described above. In some embodiments, the measurement of follistatinin culture media is used to predict which cultures of embryos to selectfor transfer contemplated to have the greatest potential to reach theblastocyst stage and beyond. Predicting embryos with greatest potentialincludes, but is not limited to, those embryos in culture whereincreased amounts of follistatin in the media is present, in comparisonto embryo cultures with lesser amount of follistatin present.

Further, embryos in culture may provide biopsies for diagnostic tests,including but not limited to genetic tests, protein tests, and proteinexpression, such as preimplantation genetic diagnosis, geneticcompatibility tests (i.e. blood type (ABO factors), Rhesus factors (Rhfactors + or −) tests, Major Histocompatibility Complex (MHC) molecules,Types I and II,) tests, etc., and mRNA expression diagnosis. Such that,in another embodiment, a diagnostic test is contemplated for therelative amount of follistatin mRNA, protein, and the like, in theembryo biopsy for estimating transplantation potential. In a furtherembodiment, a diagnostic tests comprises a test for follistatin protein,follistatin mRNA, and diagnostic tests including but not limited tothose listed herein, see, supra.

Early cleavage of human embryos to the two-cell stage afterintracytoplasmic sperm injection is an indicator of embryo viability,(for example, see, Sakkas, et al., 1998 Human Reproduction, 13:182-187;herein incorporated by reference).

Support for such diagnostic use in animals and primates, includinghumans, is provided in Patel, et al., Reproduction (2007) 133 95-106;herein incorporated by reference in its entirety, wherein microarryexperiments indicated a positive association between time of firstcleavage (oocyte competence) and follistatin mRNA abundance.Follistatin, BB, and BA subunits of inhibin/activin mRNAs weretemporally regulated during early bovine embryogenesis and peaked at the16-cell stage. Collectively, results demonstrate a positive associationof follistatin mRNA abundance with oocyte competence in two distinctmodels and dynamic regulation of follistatin, BB, and subunit mRNAs inearly embryos after initiation of transcription from the embryonicgenome. The differences in timing of first cleavage are contemplated tobe reflective of inherent differences in transcriptome composition(maternally derived transcripts) between early and late cleaving embryosat the two-cell stage.

For ART, embryos, after grading by an embryologist, are transferred intothe female uterus. Oftentimes, multiple embryos are transferred at onetime thereby improving the change of implantation and pregnancy, albeitthis can also results in multiple births if all embryos remain viableand develop.

After embryo transfer, the female typically receives progesterone shotsso that the uterus lining thickens and remains thick and suitable forimplantation. Approximately two weeks post-implantation the female ischecked for pregnancy.

Protocols change for different mammalian species; the protocol listedabove is exemplary of a human IVF protocol. However, the presentapplication is not limited to human IVF only, and IVF for othernon-human animals is equally amendable with the compositions and methodsof the present application. For example, protocol for bovine IVF can befound at, for example, Beyhan et al., 2007, Dev. Biol. 305:637-649,incorporated herein by reference in its entirety.

In some embodiments, the present invention provides nuclear transfermethods that utilize the media of the present invention. Methods fornuclear transfer cloning can be found at, for example, U.S. Pat. Nos.6,147,276, 6, 252,133, 6,525,243, 2007/0033664, and 2007/0033665 and inBeyhan et al., 2007; all incorporated herein in their entireties.Nuclear transfer techniques fall into two categories: 1) transfer of adonor nucleus (or cell containing a donor nucleus) to a maturedmetaphase II oocyte which has had its chromosomal DNA removed (e.g.,polar body removed) and 2) transfer of a donor nucleus to a fertilizedone cell zygote which has had both pronuclei removed. In ungulates theformer procedure has become the method of choice. Transfer of the donornucleus into the oocyte cytoplasm is generally achieved by inducing cellfusion. In ungulates fusion is induced by application of a DC electricalpulse across the contact/fusion plane of the couplet. The same pulsewhich induces cell fusion can also activate the recipient oocyte. Indeveloping embodiments of the present invention ionomycin was used toinduce activation of the recipient oocyte and an electrical pulse wasused to induce fusion. Following embryo reconstruction furtherdevelopment is dependent on a large number of factors including theability of the nucleus to direct development (i.e. totipotency),developmental competence of the recipient cytoplast (i.e. oocytematuration), oocyte activation, embryo culture (reviewed in Campbell andWilmut, 1994, in: Vth World Congress on Genetics as Applied to Livestock20 180-187; incorporated herein by reference in its entirety). As such,the present invention provides methods for predicting or enhancingdevelopmental competence of the recipient cytoplast.

Three methods which have been used to induce fusion are: (1) exposure ofcells to fusion-promoting chemicals, such as polyethylene glycol; (2)the use of inactivated virus, such as Sendai virus; and (3) the use ofelectrical stimulation. Exposure of cells to fusion-promoting chemicalssuch as polyethylene glycol or other glycols is a routine procedure forthe fusion of somatic cells, but it has not been widely used withembryos. As polyethylene glycol is toxic it is necessary to expose thecells for a minimum period and the need to be able to remove thechemical quickly may necessitate the removal of the zona pellucida(Kanka et al., 1991, Mol. Reprod. Dev. 29:110-116; herein incorporatedby reference). In experiments with mouse embryos, inactivated Sendaivirus provides an efficient means for the fusion of cells fromcleavage-stage embryos (Graham, 1969, Wistar Inst. Symp. Monogr. 9:19;herein incorporated by reference), with the additional experimentaladvantage that activation is not induced. In ungulates, fusion iscommonly achieved by the same electrical stimulation that is used toinduce parthenogenetic activation (Willadsen, 1986, Nature 320:63-65);Prather et al., 1987, Biol. Reprod. 37 859-866; all of which are hereinincorporated by reference). In these species, Sendai virus inducesfusion in a proportion of cases, but is not sufficiently reliable forroutine application (Willadsen, 1986, Nature 320:63-65; hereinincorporated by reference).

While cell-cell fusion is a preferred method of effecting nucleartransfer, it is not the only method that can be used. Other suitabletechniques include microinjection (Ritchie and Campbell, J. Reproductionand Fertility Abstract Series No. 15, p 60; herein incorporated byreference).

Subsequently, the fused reconstructed embryo is maintained without beingactivated so that the donor nucleus is exposed to the recipientcytoplasm for a period of time sufficient to allow the reconstructedembryo to become capable, eventually, of giving rise to a live birth(preferably of a fertile offspring).

The optimum period of time before activation varies from species tospecies. For example, for cattle, a period of from 6 to 20 hours isappropriate (e.g., activation with ionomycin for 4 minutes and culturein presence of cytocholasin B and cycloheximide for 6 h). The timeperiod should probably not be less than that which will allow chromosomeformation and it should not be so long either that the couplet activatesspontaneously or, in extreme cases that it dies. When it is time foractivation, any conventional or other suitable activation protocol isused. Recent experiments have shown that the requirements forparthenogenetic activation are more complicated. It had been assumedthat activation is an all-or-none phenomenon and that the large numberof treatments able to induce formation of a pronucleus were all causing“activation”. However, exposure of rabbit oocytes to repeated electricalpulses revealed that only selection of an appropriate series of pulsesand control of the Ca²⁺ was able to promote development of diploidizedoocytes to mid-gestation (Ozil, 1990, Development 109:117-127; hereinincorporated by reference). During fertilization there are repeated,transient increases in intracellular calcium concentration (Cutbertson &Cobbold, 1985, Nature 316:541-542; herein incorporated by reference) andelectrical pulses are believed to cause analogous increases in calciumconcentration. There is evidence that the pattern of calcium transientsvaries with species and it can be anticipated that the optimal patternof electrical pulses will vary in a similar manner. For example, theinterval between pulses in the rabbit is approximately 4 minutes (Ozil,1990, Development 109:117-127; herein incorporated by reference), and inthe mouse 10 to 20 minutes (Cutbertson & Cobbold, 1985, Nature316:541-542; herein incorporated by reference), while there arepreliminary observations in the cow that the interval is approximately20 to 30 minutes (Robl et al., 1992, in: Symposium on Cloning Mammals byNuclear Transplantation (Seidel ed.), Colorado State University, 24-27;herein incorporated by reference).

In most published experiments activation was induced with a singleelectrical pulse, but new observations suggest that the proportion ofreconstituted embryos that develop is increased by exposure to severalpulses (Collas & Robl, 1990, Biol. Reprod. 43:877-884; hereinincorporated by reference). In any individual case, a skilled artisanwill recognize that routine adjustments are made to optimize the numberof pulses, the field strength and duration of the pulses and the calciumconcentration of the medium. Other factors also contribute tocontrolling the time allowed for reprogramming, such as culture in thepresence of DMAp, cytocholasin B, and the like.

At the blastocyst stage, the embryo is screened for suitability fordevelopment to term. Typically, this is done where the embryo istransgenic and screening and selection for stable integrants has beencarried out. Screening for non-transgenic genetic markers may also becarried out at this stage. However, screening of donors at an earlierstage, as described in the present invention, are preferred.

After screening, the blastocyst embryo is allowed to develop to term.This will generally be in vivo. If development up to blastocyst hastaken place in vitro, then transfer into the final recipient animaltakes place at this stage. If blastocyst development has taken place invivo, although in principle the blastocyst can be allowed to develop toterm in the pre-blastocyst host, in practice the blastocyst will usuallybe removed from the (temporary) pre-blastocyst recipient and, afterdissection from the protective medium, will be transferred to the(permanent) post-blastocyst recipient.

I. The Use of Follistatin in the Present Inventions.

In some embodiments, the present invention provides methods for thepropagation of non-human animals, such as livestock animals. In someembodiments, the propagation of non-human animals comprising, forexample, in vitro fertilization and nuclear transfer cloning of embryos.In some embodiments, the embryos are placed in culture media comprisingfollistatin, wherein said follistatin level is at least 1 ng/ml, atleast 5 ng/ml, at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml,at least 40 ng/ml, at least 50 ng/ml, at least 100 ng/ml follistatin. Insome embodiments, maintenance and/or growth of in vitro fertilizationand nuclear transfer embryos in follistatin comprising media providesfor an increased survival of embryos in vitro and in vivo (e.g.,post-implantation) that, for example, develop into viable offspring. Insome embodiments, the present invention provides methods for generationof transgenic animals. In some embodiments, newly fertilized embryosused for transgenic gene transfer are maintained/grown in vitro infollistatin comprising media, wherein said follistatin level is at least1 ng/ml, at least 5 ng/ml, at least 10 ng/ml, at least 20 ng/ml, atleast 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 100 ng/mlfollistatin. In some embodiments, maintenance/growth of transgenicembryos in follistatin comprising media provides for an increasedsurvival of embryos in vitro and in vivo (e.g., post-implantation) that,for example, develop into viable offspring.

In humans, the causes of infertility are complex. A large proportion ofcases of infertility are attributed, at least in part, to dysfunction ofthe female reproductive system [Centers, for, Disease, Control, and,Prevention. Assisted reproductive technology success rates: nationalsummary and fertility clinic reports. In; 2001:www.cdc.gov/reproductivehealth/art.htm; herein incorporated byreference] and specifically to a reduction in oocyte quality,particularly in the case of women of advanced reproductive age[Fitzgerald, et al., Yale J Biol Med 1998; 71: 367-381; hereinincorporated by reference]. While the past decade has generally broughtadvancements in efficacy and safety of ART, there is still much room forimprovement. The most recent statistics available from the CDC indicatean overall average success rate of 29% live births from a single ARTcycle, but success rate is diminished with increased maternal age[Centers, for, Disease, Control, and, Prevention. Assisted reproductivetechnology success rates: national summary and fertility clinic reports.In; 2001: www.cdc.gov/reproductivehealth/art.htm; herein incorporated byreference, Krey, et al., Ann NY Acad Sci 2001; 943: 26-33; hereinincorporated by reference]. The tangible impact of increased maternalage on ART success cannot be dismissed, given clear societal trends fordelaying childbirth until later in life [Fitzgerald, et al., Yale J BiolMed 1998; 71: 367-381; herein incorporated by reference, te Velde,[editorial]. Maturitas 1998; 30: 103-104; herein incorporated byreference].

Furthermore, while the incidence of triplet and higher order pregnanciesresulting from ART has decreased since 1997 [Rebar, et al., N Engl J Med2004; 350: 1603-1604; herein incorporated by reference], the incidenceof singleton pregnancies has not increased [Jain, et al., N Engl J Med2004; 350: 1639-1645; herein incorporated by reference]. Multiple birthsresulted in $640 million in initial hospital costs in 2000, due toincreased infant mortality and morbidity [Hogue, Obstetrics andGynecology, 2002, 100:1017-1019; herein incorporated by reference].

Births from human ART account for greater than 30% of all twin birthsand greater than 40% of triplet and higher births [Hogue, Obstetrics andGynecology, 2002, 100: 1017-1019; herein incorporated by reference].Thus there is a need for much improvements in the efficacy and safety ofART whose success if limited by a dearth of fundamental knowledge of theintracellular and intercellular mechanisms and mediators of oocytecompetence and a lack of measurable, objective characteristicsreflective of the quality or competence of individual human oocytes attime of collection and appropriate therapeutic approaches to enhanceoocyte quality and thus embryonic development following fertilization.Elucidation of such foundational information may ultimatelyrevolutionize ART and form the basis for diagnostic approaches toincrease oocyte competence and pregnancy outcomes and (or) diagnosticapproaches to enhance embryo development and quality potentially allowfor fertilization and (or) subsequent transfer of only those embryosderived from the best quality oocytes collected and hence those with thegreatest chance for pregnancy success.

Thus the inventors contemplate the use of the present inventions to meetthe unmet need for enhancing embryonic development in vitro. Theenhancement of embryonic development of the present inventions iscontemplated to result in the increase of live births for overcominginfertility, in particular for overcoming infertility without theunwanted side effects, as described herein, such as unwanted multiplebirths.

In some embodiments, compositions and methods of the present inventionsprovide for the generation and growth of stem cells obtained fromnoncloned embryos and cloned embryos, such as for treating human diseaseand injury, for use in overcoming genetic incompatibility, providedspecific cell types for transplantation, and “embryo-friendly”derivation methods for providing (Stem Cells Vol. 24 No. 10 Oct. 2006,pp. 2162-2169; herein incorporated by reference). These potential usesare based upon the observation that these cells can, under appropriateconditions in vitro or in vivo, differentiate into most, if not all,cell types of the adult human body.

The path to successful hES (human embryonic stem cell) therapies appearstraightforward, making the desired cell type from hES cells, such asneurons to treat neurodegenerative disease or pancreatic β-cells totreat type I diabetes, and then transfer these cells to the desiredsite. However there are many difficulties in producing such cell, suchthat Embryonic stem (ES)-derived cells of the present inventions wouldalso provide a source of differenctiated cells, growth factors, andsignaling molecules for further use in stem cell therapies.

In some embodiments, compositions and methods of the present inventionsprovide for the using and maintenance noncloned embryos and cells fromthose embryos useful for therapeutic purposes including, but not limitedto, the growth of stem cells. In some embodiments, compositions andmethods of the present inventions provide for the cloning andmaintenance of cells useful for therapeutic purposes including, but notlimited to, the growth of stem cells. For example, nuclear transferembryos comprising nuclear material from a somatic cell can bemaintained/grown in follistatin comprising culture media. Growth of suchembryos, due to culture in follistatin comprising media, will exhibitincreased numbers of embryos reaching blastocyst stage, thereby,increasing the numbers of cells produced, including, but not limited to,the number of stem cells produced. As such, increased numbers of stemcells are produced per nuclear transfer embryo when applyingcompositions and methods of the present invention and more blastocyststage embryos per recipient cytoplast utilized are available forgeneration of stem cells. Stem cells produced using the compositions andmethods of the present invention find utility in, for example,therapeutics, drug discovery, and applied research.

Somatic cell nuclear transfer (SCNT) is a laboratory technique forcreating an ovum with a donor nucleus. It is used, for example, inembryonic stem cell research, or in regenerative medicine where it issometimes referred to “therapeutic cloning.” It can also be used as thefirst step in the process of reproductive cloning. In SCNT the nucleusof a somatic cell is removed and the rest of the cell discarded. At thesame time, the nucleus of an egg cell is removed. The nucleus of thesomatic cell is then inserted into the enucleated egg cell, typicallythrough a microscopic glass tube. After being inserted into the egg, thesomatic cell nucleus is reprogrammed by the host cell. The egg, nowcontaining the nucleus of a somatic cell, is stimulated with anelectrical shock and begins to divide in culture. After many mitoticdivisions in culture, this single cell forms a blastocyst with almostidentical DNA to the original organism. Inner cell mass are subsequentlyremoved, and stem cells grown out and harvested and used in, forexample, research, and/or to generate tissues and cells (therapeuticcloning).

Methods for SCNT and culture of embryos and stem cells generated can befound at, for example, Wilmut et al., 2002, Nature 419:583-586; Alberioet al., 2006, Repro. 132:709-720; Vanikar et al., 2007, Transp. Proc.39:658-661; Wakayama et al., 2001, Science, 292:740-743; allincorporated herein by reference in their entireties. Compositions andmethods of the present invention provide for developing stem cellgenerating embryos that are healthier and thereby generate more stemcells.

In some embodiments, the media of the present invention also find usefor the development and culture of embryos used to make stem cell lines,such as primate stem cell lines. Methods for obtaining pluripotent cellsfrom species in these animal orders, including monkeys, mice, rats,pigs, cattle and sheep have been previously described. See, e.g., U.S.Pat. Nos. 5,453,357; 5,523,226; 5,589,376; 5,340,740; and 5,166,065 (allof which are specifically incorporated herein by reference); as well as,Evans, et al., Theriogenology 33(1):125-128, 1990; Evans, et al.,Theriogenology 33(1):125-128, 1990; Notarianni, et al., J. Reprod.Fertil. 41(Suppl.):51-56, 1990; Giles, et al., Mol. Reprod. Dev.36:130-138, 1993; Graves, et al., Mol. Reprod. Dev. 36:424-433, 1993;Sukoyan, et al., Mol. Reprod. Dev. 33:418-431, 1992; Sukoyan, et al.,Mol. Reprod. Dev. 36:148-158, 1993; Iannaccone, et al., Dev. Biol.163:288-292, 1994; Evans & Kaufman, Nature 292:154-156, 1981; Martin,Proc Natl Acad Sci USA 78:7634-7638, 1981; Doetschman et al. Dev Biol127:224-227, 1988); Giles et al. Mol Reprod Dev 36:130-138, 1993; Graves& Moreadith, Mol Reprod Dev 36:424-433, 1993 and Bradley, et al., Nature309:255-256, 1984; all of which are herein incorporated by reference.

Primate embryonic stem cells may be preferably obtained by the methodsdisclosed in U.S. Pat. Nos. 5,843,780 and 6,200,806, each of which isincorporated herein by reference. Primate (including human) stem cellsmay also be obtained from commercial sources such as WiCell, Madison,Wis. A preferable medium for isolation of embryonic stem cells is “ESmedium.” ES medium consists of 80% Dulbecco's modified Eagle's medium(DMEM; no pyruvate, high glucose formulation, Gibco BRL), with 20% fetalbovine serum (FBS; Hyclone), 0.1 mM P-mercaptoethanol (Sigma), 1%non-essential amino acid stock (Gibco BRL). Preferably, fetal bovineserum batches are compared by testing clonal plating efficiency of a lowpassage mouse ES cell line (ES_(jt3)), a cell line developed just forthe purpose of this test. FBS batches must be compared because it hasbeen found that batches vary dramatically in their ability to supportembryonic cell growth, but any other method of assaying the competenceof FBS batches for support of embryonic cells will work as analternative.

Primate ES cells are isolated on a confluent layer of murine embryonicfibroblast in the presence of ES cell medium. Embryonic fibroblasts arepreferably obtained from 12 day old fetuses from outbred CF1 mice(SASCO), but other strains may be used as an alternative. Tissue culturedishes are preferably treated with 0.1% gelatin (type I; Sigma).Recovery of rhesus monkey embryos has been demonstrated, with recoveryof an average 0.4 to 0.6 viable embryos per rhesus monkey per month,Seshagiri et al. Am J Primatol 29:81-91, 1993; herein incorporated byreference. Embryo collection from marmoset monkey is also welldocumented (Thomson et al. “Non-surgical uterine stage preimplantationembryo collection from the common marmoset,” J Med Primatol, 23:333-336(1994); herein incorporated by reference). Here, the zona pellucida isremoved from blastocysts by brief exposure to pronase (Sigma). Forimmunosurgery, blastocysts are exposed to a 1:50 dilution of rabbitanti-marmoset spleen cell antiserum (for marmoset blastocysts) or a 1:50dilution of rabbit anti-rhesus monkey (for rhesus monkey blastocysts) inDMEM for 30 minutes, then washed for 5 minutes three times in DMEM, thenexposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3minutes.

After two further washes in DMEM, lysed trophectoderm cells are removedfrom the intact inner cell mass (ICM) by gentle pipetting, and the ICMplated on mouse inactivated (3000 rads gamma irradiation) embryonicfibroblasts. After 7-21 days, ICM-derived masses are removed fromendoderm outgrowths with a micropipette with direct observation under astereo-microscope, exposed to 0.05% Trypsin-EDTA (Gibco) supplementedwith 1% chicken serum for 3-5 minutes and gently dissociated by gentlepipetting through a flame polished micropipette.

Dissociated cells would be replated on embryonic feeder layers in freshES medium, and observed for colony formation. Colonies demonstratingES-like morphology would then be individually selected, and split againas described above. ES-like morphology is defined as compact colonieshaving a high nucleus to cytoplasm ratio and prominent nucleoli.Resulting ES cells are then routinely split by brief trypsinization orexposure to Dulbecco's Phosphate Buffered Saline (without calcium ormagnesium and with 2 mM EDTA) every 1-2 weeks as the cultures becomedense. Early passage cells would be frozen then stored in liquidnitrogen.

In one embodiment, the present invention provides compositions forincreasing embryo quality. In some embodiments, the present inventionprovides media that maintains and/or increases embryo quality after theembryos have been frozen and re-thawed. A problem in storage of embryosfor later use (e.g., for later use in in vitro fertilization or nucleartransfer) is that embryos do not tolerate freezing well and many embryosdie as a result of the storage conditions (e.g., embryos in media andliquid nitrogen freezing). The present invention provides media forembryo storage such that embryos, when stored (e.g., frozen) in media ofthe present invention are more tolerant to the freezing conditions. Assuch, the present invention provides compositions for increasing anembryo's tolerance to freezing thereby increasing the overall quality ofthe embryo.

In further embodiments, the inventors contemplate additional measures ofdetermining embryo quality, including cryotolerance, gene expressionmarkers, such as those described herein, blastomere apoptosis, etc. Assuch, the inventors contemplate the use of adding additional moleculesto medium comprising follistatin, including but not limited to anyagonists or antagonists of action of TGF beta superfamily membersproviding these molecules enhance the development and/or quality ofembryonic development and/or stem cell growth.

II. Kits.

The present invention also provides kits for determining whether a cellor tissue is expressing a follistatin gene. The present invention alsoprovides kits for adding follistatin to a cell medium. The kits areproduced in a variety of ways. In some embodiments, the kits contain atleast one reagent for specifically adding a follistatin allele orprotein. In one embodiment, the kit contains an oligonucleotide reagentfor detecting an expressed follistatin cDNA. In some embodiments, thekits contain at least one reagent for specifically adding an additionalcompound.

In some embodiments, the kits include ancillary reagents such asbuffering agents, nucleic acid stabilizing reagents, protein stabilizingreagents, and signal producing systems (e.g., florescence generatingsystems as Fret systems). The kit may be packages in any suitablemanner, typically with the elements in a single container or variouscontainers as necessary along with a sheet of instructions for carryingout the test. In some embodiments, the kits also preferably include anegative control such as a siRNA of the present inventions.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Follistatin Supplementation of IVF Embryos

The effects of follistatin treatment on in vitro produced bovine embryosduring the initial 72 h (hour) post fertilization on time to firstcleavage (at least a 2 cell embryo), development to the blastocyst stage(day 7, d7) and blastocyst cell allocation (quality) was determined.

Cumulus-oocyte complexes (COCs) were harvested from ovaries obtainedfrom a local abattoir (slaughterhouse), matured and fertilized in vitro.In vitro maturation of bovine oocytes, and in vitro fertilization andculture of embryos to the blastocyst stage were conducted as reportedpreviously (Bettegowda et al., 2006, Mol. Repro. Dev. 73:267-278; hereinincorporated by reference). After 20 h of co-incubation withspermatozoa, presumptive zygotes were stripped of cumulus cells andcultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10or 100 ng/ml Recombinant Human Follistatin (R&D Systems, Minneapolis,Minn., United States) referred to as “follistatin” in these Examples,respectively, (n=25 presumptive zygotes per treatment, n=6 replicates).

The proportion of embryos reaching the two-cell stage within 30 h (earlycleaving), 30-36 h (late cleaving) and within 48 h post fertilization(total cleavage rate) was determined. Embryos at the 8-16-cell stagewere separated 72 h after fertilization and cultured in freshpotassium-enriched simplex optimized medium (KSOM medium) supplementedwith 0.3% BSA and 10% FBS until day 7. The number of embryos reachingthe blastocyst stage at d 7 post fertilization was recorded and numbersof inner cell mass (ICM) and trophectoderm (TE) cells determined bydifferential staining.

Follistatin treatment did not increase the rate of total cleavage oraffect proportion of late cleaving embryos when compared to control.However, supplementation with 1 and 10, but not 100 ng/ml follistatinincreased the proportion of early cleaving embryos and development tothe blastocyst stage relative to controls (FIGS. 1 & 2, respectively).When early and late cleaving embryos were separated and culturedseparately, follistatin treatment (1 and 10 ng/ml) of early cleavingembryos increased (P<0.05) the rate of development to blastocyst stageand a stimulatory effect of 10 ng/ml follistatin on day 7 blastocystrate for late cleaving embryos was also observed relative to untreatedcontrols (FIG. 3). Treatment with 10 ng/ml follistatin increased(P<0.05) total cell numbers and trophectoderm cell numbers, but did notaffect numbers of inner cell mass cells relative to untreated embryos(FIG. 4). Treatment with 10 ng/ml follistatin also increased (P<0.05)proportion of trophectoderm cells and reduced the ICM/total cell ratioin day 7 blastocysts relative to controls (FIG. 5). Furthermore,treatment with 10 ng/ml follistatin increased the proportion of day 7blastocysts with a 20-40% ratio of inner cell mass to total cells(similar to in vivo produced embryos) and decreased the proportion ofday 7 blastocysts with 40-60% and >60% ICM/total cell ratios (P<0.05)(FIG. 6). Embryos with ICM ratio of 20-40% are presumed to be higherquality as >80% of in vivo-derived blastocysts were classified into thiscategory in previous studies (Koo et al., 2002, Biol. Repro. 67:487-492;herein incorporated by reference).

Example 2 siRNA Mediated Knockdown of Follistatin mRNA and its Effect onDevelopment of IVF Embryos

To determine the need for endogenous follistatin for early embryonicdevelopment, the effect of follistatin RNA knockdown (e.g., viamicroinjection of follistatin siRNA) on time to first cleavage andsuccessful development of in vitro fertilized bovine embryos to the 8-16cell and blastocyst stages were determined. After 20 h of co-incubationwith spermatozoa, presumptive zygotes were stripped of cumulus cells andcultured in KSOM medium supplemented with 0.3% BSA and microinjectedwith follistatin siRNA (25 μM). Presumptive zygotes injected withsimilar concentration of negative control siRNA (Ambion UniversalControl #1) or water (sham microinjection and uninjected 1 embryos allserved as controls).

Follistatin siRNA injection (25 μM) into presumptive zygotes decreasedamounts of follistatin mRNA at the 4-cell stage (FIG. 7) byapproximately 80% relative to uninjected, sham injected and negativecontrol siRNA injected embryos (P<0.05), and dramatically reducedfollistatin protein in 16-cell embryos (determined byimmunofluorescence) relative to uninjected controls demonstratingefficacy and specificity of follistatin siRNA in reducing follistatinmRNA and protein abundance in early bovine embryos (FIG. 7).Microinjection of follistatin siRNA into presumptive zygotes did notinfluence proportion of embryos cleaving early (within 30 h) but reducedproportion of embryos developing to the 8-16 cell and blastocystrelative to uninjected, sham injected and negative control siRNAinjected embryos and proportion of day 7 blastocysts (FIG. 8). As such,experimental results as found in FIGS. 1-8 provide evidence for a roleof follistatin in early embryogenesis.

Example 3 Follistatin Supplementation Effects on the Development ofNuclear Transfer Embryos

To demonstrate that follistatin treatment enhances the efficiency ofnuclear transfer, the effects of follistatin supplementation on cellallocation and blastocyst development of nuclear transfer embryos wasdetermined. The generation of nuclear transfer embryos was conducted asdescribed by Beyhan et al., 2007, Dev. Biol. 305:637-649; hereinincorporated by reference. After nuclear transfer, activation andfusion, embryos were cultured as described in Example 2 (n=4 replicatesof 25 embryos per treatment) in the presence of 0, 1, 10 and 100 ng/mlof follistatin. Untreated parthenogenetic embryos were included asquality control for oocytes and the activation procedure. The numbers oftrophectoderm (TE) versus inner cell mass (ICM) cells and total cellnumbers were determined by cell counts on differentially stained embryos(Machaty et al., 1998, Biol. Repro. 59:451-455; herein incorporated byreference).

Due to defects in placentation and blastocyst cell allocationcharacteristic of nuclear transfer embryos (presumably leading to highrates of pregnancy loss), this experiment was conducted to determine theeffects of follistatin supplementation on development of nucleartransfer embryos to the blastocyst stage and on blastocyst cellallocation. Treatment with 10 ng/ml follistatin significantly increasedthe proportion of nuclear transfer embryos developing to the blastocyststage relative to untreated nuclear transfer embryos (P<0.05) (FIG. 9).While not significantly affecting numbers of ICM and total cells,treatment with 10 ng/ml follistatin significantly increased numbers oftrophectoderm cells and proportion of trophectoderm cells to total cellsand decreased the proportion of inner cell mass cells to total cellsrelative to untreated nuclear transfer embryos (P<0.05) (FIG. 10). Nosignificant effect of other doses of follistatin was observed for theabove endpoints. However, treatment with 1 and 10 ng/ml follistatinreduced the proportion of day 7 nuclear transfer blastocysts with an ICMratio of >60% relative to untreated nuclear transfer blastocysts (FIG.11). No significant effect of follistatin treatment on proportions ofday 7 nuclear transfer blastocysts with ICM ratio of 20-40% or 40-60%was observed (FIG. 12).

Collectively, the results of the above studies demonstrate thatexogenous follistatin treatment during the early stages of in vitrobovine embryo development enhances time to first cleavage, developmentto the blastocyst stage and cell allocation in favor of increasedtrophectoderm cells, thereby supporting a functional requirement offollistatin for early embryonic development. The results also indicatethat follistatin treatment can be used to increase development ofnuclear transfer embryos to the blastocyst stage and enhance blastocystcell allocation in favor of trophectoderm cells for enhancingplacentation and ultimately birth of live offspring.

Example 4 This Example Demonstrates an Exemplary Follistatin Specific onEmbryonic Development

Specifically, a negative effect of siRNA mediated knockdown offollistatin mRNA was demonstrated using in vitro fertilized embryos andcapability of exogenous follistatin to rescue embryonic development.

Experiments such as those described in Example-2 (FIG. 8) were initiatedfor inhibiting endogenous follistatin expression and production. As anadditional experimental condition endogenous follistatin was loweredusing the siRNA construct in the presence of exogenous follistatin at 10ng/ml in the culture medium. Exogenous follistatin. Rescued the percent8-16 cell embryos at 72 hr, and recovered the percent in blastocytestage (FIG. 13). Further, exogenous follistatin showed little effect asdid the silencing construct on the number of cells within the inner cellmass (ICM), while recovering the relative number of lost trophectoderm(TE) cells and recovering total cell numbers (TOTAL) in culture (FIG.14).

Thus, the exogenously added follistatin can enhance embryo qualitydemonstrated the recovery of TE cell numbers.

Example 5 This Example Demonstrates an Exemplary Follistatin SpecificNegative Effect of Follistatin siRNA on TE Cell Numbers (a Marker ofEmbryo Quality) and Further Ablation Replacement Effects on BlastocystmRNA Abundance for CDX2 (a Trophectoderm Cell Marker) and Lack of Effecton Inner Cell Mass (ICM) Cell Marker (Nanog)

Experiments such as those described in Example-4 (FIGS. 13 and 14) wereinitiated for determining relative RNA abundance of molecules associatedwith transplantation potential.

Recent research applied to the mouse has established that TE and ICMdifferentially express several lineage-specific transcription factors.Cdx2 becomes restricted to the TE and is required for TE formation(Yamanaka et al. 2006, Cell 126:663-676; FIG. 2). In contrast, Oct4 andNanog become restricted to and influence ICM fate (Yamanaka et al. 2006,Cell 126:663-676; FIG. 2). The current understanding of their roles ledto a model that predicts mutual antagonism between Oct4 and Cdx2 insupporting the formation of TE and ICM fates in the blastocyst (Yamanakaet al. 2006, Cell 126:663-676). Further, cdx2 is associated withsuccessful (relatively higher) transplantation potential(Chawengsaksophak, et al., Proc Natl Acad Sci USA 2004; 101:7641-7645;Strumpf, et al. Development 2005; 132:2093-2102; Meissner, et al. Nature2006; 439:212-215; each of which is herein incorporated by reference).

Nanog is a homeodomain transcription factor that is expressedspecifically in undifferentiated embryonic stem (ES) cells and was shownto be essential in the maintenance of pluripotency in mouse ES cells.Knockdown experiments using NANOG small interfering (si) RNA resulted ininduction of differentiation markers such as AFP, GATA4 and GATA6 forthe endoderm and CDX2 for the trophectoderm. These results suggest thatNANOG plays a crucial role in maintaining the pluripotent state ofprimate ES cells. NANOG over-expressing cell lines retained theirundifferentiated state in the absence of a feeder layer, as shown byexpression of undifferentiated ES cell markers such as alkalinephosphatase (ALP) and OCT-4 (Yasuda, et al., Genes Cells. 2006September; 11(9):1115-23; herein incorporated by reference).

Further, biopsies of embryos that resulted in calf delivery wereenriched with genes necessary for implantation (COX2 and CDX2),carbohydrate metabolism (ALOX15), growth factor (BMP15), signaltransduction (PLAU), and placenta-specific 8 (PLAC8). Biopsies fromembryos resulting in resorption are enriched with transcripts involvedprotein phosphorylation (KRT8), plasma membrane (OCLN), and glucosemetabolism (PGK1 and AKR1B1). Biopsies from embryos resulting in nopregnancy are enriched with transcripts involved inflammatory cytokines(TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1,PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is aninhibitor of implantation.

The caudal-related homeobox protein CDX2 is a transcriptional regulatoressential for trophoblast lineage, functioning as early as implantation.The CDX2 gene is the earliest trophoblast-specific transcription factorreported to date (Roberts, et al., Reprod Biol Endocrinol 2: 47, 2004,Tolkunova, et al., Stem Cells 24: 139-144, 2006; each of which areherein incorporated by reference). An earlier gene targeting approachdemonstrated that CDX2 null embryos fail to implant, suggestive of amajor defect in TE development (Chawengsaksophak, et al., Nature386:84-87, 1997, Rossant, Stem Cells, 19:477-482, 2001; each of whichare herein incorporated by reference). The same authors showed that theimplantation failure was due to loss of TE epithelial integrity and/orincreased incidence of apoptosis of TE cells.

Therefore, CDX2 is one of the genes crucial for placental development,by which its aberrations in embryo can result in implantation orplacental defect as reported by Hall et al. (Hall, et al., Reprod FertilDev 17: 261-261, 2005). See, Ashraf El-Sayed, et al., Physiol Genomics28:84-96, 2006. First published Oct. 3, 2006; herein incorporated byreference.

Quantification of Oct-4 and CDX-2 mRNA in Bovine Blastocysts:

RNA isolation and quantitative real-time RT-PCR analysis of mRNAabundance for Nanog and CDX-2 was conducted according to proceduresdescribed in Bettegowda, et al., 2006, Mol Reprod Dev 73:267-278;Bettegowda, et al., 2007, Proceedings of the National Academy ofSciences USA 104:17602-17607; and Bettegowda, et al., 2008, Biology ofReproduction DOI:10.1095/biolreprod.107.067223; each of which are hereinincorporated by reference. Briefly, total RNA was extracted from each ofthe blastocyst samples (n=4 pools of 2 blastocysts per pool for eachtreatment) using the RNAqueous micro kit (Ambion) according tomanufacturer's instructions. The RNA was eluted twice using a 10 μlvolume of prewarmed (75° C.) elution solution according tomanufacturer's instructions. Residual genomic DNA in all extractedsamples was removed by DNAse I digestion (Ambion). Total RNA (10 μl)from each sample for real-time RT-PCR analysis was utilized for reversetranscription (RT) using oligo dT₍₁₅₎ primers as described elsewhere.Embryos were evaluated for cleavage (Bettegowda et al., 2006, Mol ReprodDev 73:267-278; herein incorporated by reference). After termination ofcDNA synthesis, each RT reaction was then diluted with nuclease freewater (Ambion) to a final volume of 100 μl. The quantification of allgene transcripts (Nanog, CDX2 and 18S rRNA) was done by real-timequantitative RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems,Foster City, Calif.). Primers were designed for use using the PrimerExpress program (Applied Biosystems) and derived from bovine sequencesfound in GenBank (see Table 1). A primer matrix was performed for eachgene tested to determine the optimal primer concentrations. Eachreaction mixture consisted of 2 μl cDNA, 1.5 μl each of forward (5 μM)and reverse primers (5 μM), 7.5 μl nuclease-free water, and 12.5 μl SYBRGreen PCR Master Mix in a total reaction volume of 25 μl (96-wellplates). Reactions were performed in duplicate for each sample in an ABIprism 7000 Sequence Detection System (Applied Biosystems). For real-timeRT-PCR experiments, the amounts of mRNAs of interest (CDX-2, Nanog) werenormalized relative to abundance of an endogenous control (18S rRNA) toaccount for differences in total RNA concentrations between samples. Themean sample threshold cycle (CT) and mean endogenous control CT for eachsample were calculated from duplicate wells. The relative amounts oftarget gene expression for each sample were calculated by using theformula 2—(ΔΔCT) as described elsewhere (Livak, et al., Methods 2001;25:402-408; herein incorporated by reference). Effects of treatments onblastocyst mRNA abundance for CDX2 and Nanog was determined by one-wayanalysis of variance followed by Fisher's protected least significantdifference test.

TABLE 1 Genebank Accession Gene number PCR Primer Sequence CDX-2AM293662 F: 5′-FCGTCTGGAGCTGGAGAAGGA-3′ R: 5′-CGGCCAGTTCGGCTTTC-3′ NanogNM 001025344 F: 5′-AAAGTTACGTGTCCTTGCAAACG-3′ R:5′-GAGGAGGGAAGAGGAGAGACAGT-3′

Example 6 Effect of Follistatin Treatment on Rhesus Monkey EmbryonicDevelopment

The inventors discovered that follistatin supplementation enhances thedevelopment of nuclear transfer embryos of primates.

Methods:

In vitro fertilization (IVF) of rhesus monkey oocytes and culture ofembryos was conducted according to previously described procedures(Vandevoort et al., J In Vitro Fert Embryo Transf 1989; 6: 85-91;VandeVoort et al., Theriogenology 2003; 59: 699-707; each of which areherein incorporated by reference).

After IVF, presumptive zygotes were cultured in Hamster embryo culturemedium 9 plus PVA (HECM-9 PVA) for 48 h post insemination in thepresence or absence of 10 ng/ml follistatin (n=6 replicates and 86 and72 embryos total for control and follistatin treatments. Embryos weresubsequently cultured in HECM-9 PVA containing 5% bovine calf serum (inthe absence of exogenous follistatin).

Embryos were evaluated for cleavage at 30 hr. post inseminationincluding counting the number of cleaved embryos and evaluated forblastocyte development at 48 and 188 hours post insemination (hpi).

The percent of embryos in culture which had cleaved by 30 h postinsemination (hpi) were determined and further percent embryos reachingthe blastocyst stage at 48 hpi and blastocyst development atapproximately 188 hpi were determined.

The effect of follistatin treatment on % cleavage at 30 hpi and %blastocyst development was determined statistically by t test analysis.

As shown in FIG. 16, follistatin supplementation significantly enhancedthe development of rhesus monkey embryos. The progression of developmentof fertilized embryos to the M2 stage following cleavage wassignificantly increased (FIG. 16A). Further, the progression ofdevelopment to the blastocyst stage was also significantly increased(FIG. 16B).

Therefore the inventors contemplate the use of follistatinsupplementation in culture media for enhancing assisted reproductivetechniques and stem cell development and further for using measurementsof follistatin expression in diagnostic tests, as described herein,specifically for primates including humans.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

1. A culture medium for in vitro culture of embryos comprisingfollistatin, wherein the development and survival of said embryos isenhanced when grown in said culture media compared to growth in culturemedia without follistatin.
 2. The culture medium of claim 1, whereinsaid follistatin is present at a concentration from about 1 ng/ml toabout 20 ng/ml.
 3. The culture medium of claim 1, wherein saidfollistatin is present at a concentration of about 10 ng/ml.
 4. Theculture medium of claim 1, further comprising an embryo, wherein saidembryo is selected from the group consisting of in vitro fertilizationembryos, nuclear transfer embryos, cloned embryos, noncloned embryos,embryos for assisted reproductive techniques.
 5. The culture medium ofclaim 1, wherein said embryos are mammalian embryos.
 6. A kit comprisingthe culture medium of claim
 5. 7. A method of increasing the survival ofembryos, comprising, a) providing, i) an embryo, ii) a culture medium,wherein said culture medium comprises follistatin, b) culturing saidembryo in a culture medium wherein the survival of said embryo isincreased compared to survival of an embryo not grown in said media. 8.The method of claim 7, wherein said embryo is an in vitro fertilizationembryo.
 9. The method of claim 7, wherein said nuclear transfer embryois a nuclear transfer embryo.
 10. The method of claim 7, wherein saidnuclear transfer embryo comprises genetic material obtained from asomatic cell.
 11. The method of claim 7, wherein said follistatin ispresent at a concentration from about 1 ng/ml to about 20 ng/ml.
 12. Themethod of claim 7, wherein said follistatin is present at aconcentration of about 10 ng/ml.
 13. The method of claim 7, whereinincreased survival of said embryo is increasing the number oftrophectoderm cells.
 14. The method of claim 7, wherein said follistatinis a human recombinant follistatin.
 15. The method of claim 7, whereinsaid method further comprises obtaining a biopsy from said embryo. 16.The method of claim 15, wherein said method further comprises c)determining the amount of follistatin expression in said embryo.
 17. Amethod of generating stem cells comprising: a) providing, i) an embryo,ii) a culture medium, wherein said culture medium comprises follistatin,b) culturing said embryo in a culture medium to provide culturedembryonic cells, c) generating stem cells from said cultured embryoniccells.
 18. The method of claim 17, wherein culture of said embryo insaid media generates an increased number of stem cells from said embryoas compared to the number of stem cells generated from an embryo notgrown in said culture medium.
 19. The method of claim 17, furthercomprising step d) harvesting said stem cells.
 20. The method of claim17, wherein said follistatin is present at a concentration from about 1ng/ml to about 20 ng/ml.